AREA 1. MEMBRANE BIOENERGETICS, TRANSPORT AND SIGNALING Two classes of membrane channels expressed in sensory neurons were the focus in Area 2. First, the family of P2X receptor channels, which are activated by extracellular ATP and serve important roles in nociception and sensory hypersensitization. Second, TRPV channels, play a central role in thermosensation and the generation of pain signals. Both classes of channels are potential therapeutic targets and of major biomedical significance. The first project was a collaboration with Dr. Kenton J. Swartz at NINDS and Prof. Motoyuki Hattori from Fudan University (China), and focused on P2X3 and its interplay with ATP and Mg2+ (Li et al. eLife 2019). This project integrated single-particle cryo-EM studies of P2X3-ATP-Mg2+ complexes, a comprehensive functional characterization through electrophysiological recordings, and molecular dynamics simulations. Our results revealed a previously unrecognized structural element in P2X3 receptors that is essential for channel activation under physiological conditions where the predominant form of ATP available is bound to Mg2+. Our results also revealed that Mg2+ ions play a fundamental role in tuning the gating properties of this class of ATP-activated receptor-channels. Specifically, following the activation and desensitization of the channel, Mg2+ hampers ATP dissociation, acting cooperatively with the protein, to slow down the recovery from desensitization. In doing so, Mg2+ reducing the frequency with which the channel is available for activation by extracellular ATP, i.e. it precludes triggering of new sensory signals. In the second project, we initiated a collaboration with Prof. Valeria Vasquez at the University of Tennessee and Prof. Avi Priel at the Hebrew University of Jerusalem (Israel), in which aimed to gain structural insights into the mode by which TRPV1 channels become activated when exposed to toxins from venomous organisms, thus triggering pain signals (Geron et al. PNAS 2018). This collaborative study examined wildtype and mutagenized rat TRPV1 (rTRPV1) through electrophysiological recordings (whole-cell and single-channel), behavioral analysis in a transgenic Caenohrabditis elegans expressing rTRPV1, and computational structural modeling. The study focused on the effect of the double-knot tarantula toxin, DkTx, which binds on the extracellular surface of the channel. Our results demonstrated that a feature of the TRPV1 structure known as the pore turret modulates the interaction of the channel with DkTx, and thereby contributes to dictate different gating modalities in response to intracellular agonists acting through binding domains on the other side of the membrane, such as capsaicin. This study underscores the remarkable allosteric mechanisms of this class of channels, which permit them to integrate diverse stimuli from multiple sources and thereby evoke an appropriate pain responses. AREA 2. DEVELOPMENT OF SIMULATION METHODS The central achievement in Area 3 has been the development and release of a novel molecular-simulation method to calculate the free energy of a shape transformation in a lipid membrane directly from a molecular dynamics simulation. In this method, the bilayer need not be homogeneous or symmetric and can be atomically-detailed or coarse-grained. The methodology is based on a collective variable that quantifies the similarity between the membrane and a set of pre-defined 3D density distributions. Enhanced sampling of this variable, which we refer to as Multi-Map, re-shapes the bilayer and permits derivation of the corresponding potential-of-mean-force. Calculated energies thus reflect the dynamic interplay of atoms and molecules, rather than postulated effects based on pre-conceived theories or mathematical models. In the first publication based on this method (Fiorin et al. J Comput Chem 2019), we evaluated a series of membranes deformations of different shape, amplitude, range and composition. These studies demonstrated that the macroscopic bending modulus assumed by standard theories, such as the Helfrich-Canham model, is increasingly unsuitable as the scale of the deformation narrows below the 100- range. This is worth noting as it is in this length-scale that many important processes in membrane biology occur, most notably the regulation of membrane protein structure and function by lipids. In this range direct free-energy calculations based on the Multi-Map method reveal a much greater plasticity that conventional theories. We also quantified the stiffening effect of cholesterol on bilayers of different composition, and compared our results with experiments, successfully. Lastly, we illustrated how our approach facilitates analysis of other solvent reorganization processes, by examining the energetics of wetting and de-wetting of a hydrophobic pore. We posit that this approach will foster a range of advancements in fundamental and applied membrane biophysics research. On a fundamental level, we have illustrated how the method can be used to evaluate deformations beyond the limits of the Helfrich-Canham model, and thus serve as a means to calibrate semi-empirical models of membrane energetics. A range of other questions, such as how lipid composition impacts rigidity and spontaneous curvature, will be similarly amenable. We also anticipate that the Multi-Map collective variable will facilitate the characterization of the interplay between membrane morphology and membrane protein structure and mechanisms, and that it will enable quantitative studies of solvent fluctuations in multicomponent systems and irregular geometries.